The relationship of ALPK1, hyaluronic acid and M1 macrophage polarization in the temporomandibular joint synovitis

Abstract M1 macrophage polarization and synovitis play an important role in the pathogenesis of temporomandibular joint osteoarthritis (TMJOA). Reduced molecular weight of hyaluronic acid (HA) in synovial fluid of patients with TMJOA. In addition, high molecular weight hyaluronic acid (HMW‐HA) is often used clinically to treat TMJ inflammation. As a pattern recognition receptor of the cytoplasm, ALPK1 was found to be pro‐inflammatory in a variety of diseases. However, the relationship of ALPK1, HA and M1 macrophage polarization in TMJ synovitis remains unclear. We aimed to investigate the role of ALPK1 and HA in macrophage polarization and TMJ synovitis and the underlying mechanisms. The results demonstrated that ALPK1 was highly upregulated in the synovial macrophages in the inflamed TMJ synovium of patients. Low molecular weight hyaluronic acid (LMW‐HA) promoted the expression of ALPK1 and M1 macrophage‐associated genes. Besides, rhALPK1 promoted the expression of M1 macrophage‐associated factors and the nuclear translocation of PKM2. Furthermore, ALPK1 knockout mice exhibited limited infiltration of macrophages and decreased expression levels of M1 macrophage‐associated genes in CFA‐induced TMJ synovitis. While HMW‐HA inhibited the expression of ALPK1 and M1 macrophage polarization. Our results elucidated that ALPK1 promoted TMJ synovitis by promoting nuclear PKM2‐mediated M1 macrophage polarization, whereas HMW‐HA inhibited the expression of ALPK1 as well as M1 macrophage polarization.

M1 polarization of synovial macrophages aggravates CFA-induced experimental TMJ inflammation by producing a large number of proinflammatory cytokines including IL-1β, IL-6 and TNFα. 8,9PK1 is a pattern recognition receptor of cytoplasm for ADPheptose. 10 A series of studies have demonstrated that ALPK1 plays an important role in the occurrence and development of various inflammatory diseases.11,12 ALPK1 could promote the expression of TNFα and IL-8, and inhibiting ALPK1 could effectively inhibit inflammation.13,14 A study found that the expression of ALPK1 was positively correlated with macrophage infiltration in renal tissue. 15Faass, L. et al. found that pure ADP-heptose could promote the activation of THP-1(a human acute monocytic leukaemia cell line) cells and human primary macrophages, while ADP-heptose and bacterial enzyme-treated lysates could promote the differentiation of monocytes into M1 macrophages.16 Concerning that ADP-heptose functions following the combination of ALPK1, 17 it is reasonable to hypothesize that ALPK1 may regulate M1 macrophage polarization to participate in synovial inflammation of TMJ.
PKM2 is a key rate-limiting enzyme for glycolysis.Recent studies have shown that PKM2 plays an important role in the M1 macrophage polarization. 18The monomers and dimers of PKM2 can enter the nucleus to promote glycolysis and inflammatory responses, and the tetramer of PKM2 in the cytoplasm can mediate the tricarboxylic acid cycle, oxidative phosphorylation and anti-inflammatory responses. 19 is a normal component of TMJ synovial fluid, which has important effects such as lubrication and anti-inflammation. 20Decreased molecular weight of HA has been found in the synovial fluid of patients with temporomandibular joint (TMJ) disorders. 21Studies have shown a significant correlation between the molecular weight of HA and its biological effects. 22,23LMW-HA promotes M1 macrophage polarization, 24 while HMW-HA attenuates synovitis by promoting M2 macrophage polarization. 25It has been demonstrated that HMW-HA is effective in the treatment of TMJ inflammation in animal experiments 26 and clinical. 27However, the relationship of HA, ALPK1 and macrophage polarization in TMJ synovitis remains unclear.
In the present study, the alteration of ALPK1 in the inflamed synovium and synovial fluid of human TMJs was analysed.Subsequently, a CFA-induced TMJ synovitis model and ALPK1 knockout mice were used to investigate the function of ALPK1 in synovial inflammation as well as M1 macrophage polarization.Moreover, the role of ALPK1 and HA in M1 macrophage polarization was explored with the RAW264.7 cell line.Our results suggested that ALPK1 played an important role in the pathogenesis of TMJ synovitis and provided valuable insights for the treatment of TMJOA with HMW-HA.

| Clinical samples
Synovial fluid samples were collected from 11 patients with clinically confirmed TMJ synovitis and 8 patients suffered from TMJ anterior disc displacement without synovitis.Specimens of inflamed synovium were obtained from five patients with TMJOA who were observed with obvious TMJ synovitis under direct vision in arthroplasty.The control synovial tissues were derived from five patients with condylar hyperplasia.All of the experiment patients were on the premise of informed consent.The ethics approval was granted by the Ethics Committee of Wuhan University Hospital of Stomatology (approval No. 2019-A15).All methods were carried out in accordance with relevant guidelines and regulations.

| Animal model
Eight-week-old C57/BL6 male wild-type (WT) mice and ALPK1 −/− mice were employed in this experiment.ALPK1 knockout mice were produced as previously described. 28Synovial inflammation was induced by bilateral injection of 20 μL CFA (F5881; Sigma) into the anterosuperior compartment of TMJ, and the control group received isopycnic saline.Specimens from WT and ALPK1 −/− mice were sacrificed 1 or 2 weeks after CFA injection.All animal studies were approved by the Ethics Committee for Animal Research, School and Hospital of Stomatology, Wuhan University (approval No. S07919110A), were operated in accordance with national guidelines for the housing and care of laboratory animals, and conformed to the ARRIVE 2.0 guidelines.

| Histologic staining
TMJ samples were collected, using 4% paraformaldehyde and 10% EDTA for fixation and decalcification, and following embedded in paraffin, continuous mid-sagittal sections of 4 μm were cut for subsequent staining.After dewaxing and gradient hydration, haematoxylin and eosin (HE) staining was performed to quantify the synovial inflammation.
Immunofluorescence staining was incubated with the designated primary antibody overnight at 4°C and then conjugated the primary antibody with a fluorescent secondary antibody to visualize staining, and 4′,6-diamidino-2-phenylindole (DAPI) reagent was used to develop nuclei.Primary antibodies were as follows: Immunohistochemical staining was to incubate with the designated primary antibody at 4°C overnight, then with the designated secondary antibody for 30 min, and finally developed the colour with DAB (DAB-0031, Bio technologies), followed by counterstaining of nuclei with haematoxylin.Primary antibodies were as follows: rabbit anti-TNFα (1:400, 11948S, Cell Signaling Technology), rabbit anti-IL-1β (1:400, YT2322, Immunoway) and rabbit anti-IL-6 (1:400, A14687, ABclonal).

| Cell viability assay with CCK-8
Cells were seeded in 96-well plates at a density of 5 × 10 4 per well.
Then, the cells were treated with rhALPK1 at concentrations of 0, 2, 5, 10, 15, 20 and 25 ng/mL, and the cells were stored in a 37°C, 5% CO 2 incubator.After 24 h, 200 μL of 10% CCK-8 solution diluted in DMEM medium was add to each well.After further incubation for 2 h, use a microplate reader to measure the absorbance of each well at a wavelength of 450 nm.

| Enzyme-linked immunosorbent assay (ELISA)
The synovial fluid and cell supernatant were tested according to the protocols of the ELISA kit (Human ALPK1 ELISA Kit, JL14242, Jianglai Biotechnology; Mouse IL-1β ELISA Kit, CME0006, Sizhengbai Biotechnology; Mouse TNFα ELISA Kit, CME0004, Sizhengbai Biotechnology).In addition, the relative concentration of ALPK1 in synovial fluid is calibrated using the total protein concentration measured by BCA.

| Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted with Trizol, and then, 1ug RNA was reversely transcribed to cDNA according to the manufacturer's protocol (Takara).The qRT-PCR reactions were performed on the Biorad CFXTM Connect Real-Time System according to the manufacturer's protocol (Takara), as described previously. 29

| Statistical analysis
Statistical analyses were conducted in Prism 8.0 (GraphPad prism8), and p < 0.05 was considered statistically significant.Comparisons between two groups were performed with Student's t-test.
Comparisons between three and six groups were performed with one-way analysis of variance.Notably, human data were considered nonparametric distributions and comparisons between two independent groups were analysed by Mann-Whitney U-test.

| ALPK1 is highly expressed in macrophages of TMJ inflamed synovial fluid and synovium
First, upregulation of ALPK1 was discovered in synovial fluid from patients with TMJ synovitis in comparison with patients with TMJ anterior disc displacement by ELISA analysis (Figure 1A). Figure 1B showed the control synovium and inflamed synovium of TMJ by HE staining.
The immunofluorescence analysis showed that the expression of ALPK1 in inflamed synovium was higher than that in control synovium (Figure 1C,D).In addition, synovial macrophages in the inflamed synovium showed abundant expression of ALPK1, whereas expression by synovial fibroblast was barely detectable (Figure 1E,F).These results suggested that a high level of ALPK1 was discovered in macrophages of inflamed synovium and may be involved in TMJ synovitis.

| LMW-HA enhances the expression of ALPK1 and M1 macrophage polarization
Reduced molecular weight of HA has been found in the pathogenesis of TMJOA. 21So, it aroused our interest that what is the effect of LMW-HA on ALPK1, a possible synovitis-associated molecule in macrophages.In our results, western blot analysis revealed that ALPK1, as well as the inflammatory factors INOS and TNFα, are elevated under stimulation by LMW-HA (Figure 2A,C).qPCR assay showed that the expression of ALPK1 and M1 macrophageassociated genes INOS, TNFα, IL-6 and IL-1β was increased under treatment with LMW-HA (Figure 2B,D).Our study showed that the pro-inflammatory substance LMW-HA promoted the expression of ALPK1, further suggesting that ALPK1 may act as a proinflammatory gene in TMJ synovitis.

| ALPK1 promotes M1 macrophage polarization in vitro
To study the role of ALPK1 in macrophages, we determined the optimal concentration of rhALPK1 in RAW264.cells by qPCR analysis (Figure 3A).Consistently, protein analysis results showed that ALPK1 could promote the expression of INOS and IL-1β (Figure 3B,C).Meanwhile, ALPK1 promoted the secretion of inflammatory factors including TNFα and IL-6 (Figure 3D).
Consistently, immunofluorescence showed that ALPK1 promoted the expression of INOS (Figure 3E,F).Taken together, these findings suggested that ALPK1 promoted M1 macrophage polarization in RAW264.7 cells.

| ALPK1 deficiency alleviates CFA-induced synovial inflammation in TMJ of mice
To further investigate the role of ALPK1 in TMJ synovitis, we established an ALPK1 knockout mice.In the current study, TMJ synovitis mice were employed and harvested 1 and 2 weeks after CFA injection.In contrast to WT mice, ALPK1 −/− mice exhibited decreased synovial cell layers, stromal cell density and inflammatory cell infiltration by HE staining (Figure 4A), as well as significantly lower synovitis scores (Figure 4C).We further

| ALPK1 promotes M1 macrophage polarization by increasing the nuclear translocation of PKM2
Numerous studies have shown that nuclear PKM2 plays an important role in M1 macrophage polarization. 30,31However, whether PKM2 mediates ALPK1-promoted M1 macrophage polarization remains to be explored.In our study, as shown in Figure 6A,B, PKM2 nuclear translocation was increased, but PKM2 in cytosolic had no significant change in RAW264.7 cells stimulated by rhALPK1.To further explore the role of PKM2 in ALPK1 promoting M1 macrophage polarization, DASA-58, which is a specific potent activator of PKM2 and can prevent PKM2 nuclear translocation, 30,31  Similarly, the secretion of TNF-α and IL-6 was reversed by DASA-58 (Figure 6F).Collectively, these findings demonstrated that ALPK1 promoted M1 macrophage polarization by increasing nuclear translocation of PKM2.

| HMW-HA inhibits the expression of ALPK1 and M1 macrophage polarization
HMW-HA is often used for the treatment of TMJ inflammation. 27e impact of HMW-HA on the expression of ALPK1 and the activation of macrophages has attracted our attention.In this study, RAW264.Taken together, these findings suggested that HMW-HA had an anti-inflammatory effect and inhibited the expression of ALPK1 and M1 macrophage polarization.

| DISCUSS ION
Recent studies have demonstrated that ALPK1 is expressed in the testes of mice, 32 kidneys of humans 33 and intestinal mucosa of humans. 34In the present study, it was found that ALPK1 was highly expressed in the synovial fluid and inflamed synovium of TMJ patients.Macrophages of inflamed synovium showed abundant expression of ALPK1, whereas expression by synovial fibroblasts was barely detectable.The possible reason is that certain factors are highly expressed in macrophages in the specific environment.Similarly, Kong, Q. et al. found that expression of HSPA12A in liver macrophages was higher than that in Hepatocytes. 35Intriguingly, although ALPK1 was preferably expressed in synovial macrophages, there is some positive staining of ALPK1 in a few synovial fibroblasts, which may be due to their cluster heterogeneity. 36 present, studies have confirmed that ALPK1 is a promoting factor in several inflammation-related diseases. 11,12Meanwhile, M1 macrophage polarization plays an important role in the development of joint inflammation. 37Moreover, M1 macrophages play a more critical role in cartilage damage in TMJOA patients than other common cell populations. 38Inhibition of M1 macrophage polarization alleviates TMJOA. 39The molecular weight of HA decreases in TMJOA, 21 and many studies have suggested that LMW-HA promoted M1 macrophage polarization. 40,41In our experiments, we found that LMW-HA not only promoted M1 macrophage polarization but also the expression of ALPK1.Furthermore, rhALPK1 promoted the expression of M1 macrophage markers CD86 and INOS, and the expression of inflammatory cytokines TNFα, IL-1β and IL-6 in RAW264.7 cells.ALPK1 knockout mice and CFA-induced TMJ synovitis model were used for further studies.Our study found that ALPK1 knockout mice exhibited decreased synovial cell layers, stromal cell density and inflammatory cell infiltration, as well as significantly lower synovitis scores, indicating that ALPK1 could promote the development of TMJ synovitis.These results suggested that ALPK1 aggravated synovitis by promoting M1 macrophage polarization.
Glycolysis plays an important role in M1 macrophage polarization. 42In M1 macrophages, there is a Warburg effect similar to that observed in tumour cells. 43The Warburg effect means that in the presence of a lot of oxygen, glucose is still converted into lactate, that is, cells produce energy through glycolysis. 44As a key ratelimiting enzyme for glycolysis, PKM2 has been shown an important role in the M1 macrophage polarization. 18In this study, we demonstrated that ALPK1 could promote nuclear translocation of PKM2 in macrophages.Importantly, DASA-58 significantly reversed the enhanced production of INOS, CD86 and IL-1β, and secretion of IL-6 and TNFα in these cells induced by rhALPK1, demonstrating that ALPK1 regulates synovitis by promoting nuclear PKM2-mediated M1 macrophage polarization.
As a normal component of the TMJ synovial fluid, HMW-HA is widely used clinically in the treatment of TMJ inflammation. 27In recent years, HA-related biological materials have also shown good therapeutic effects in TMJ inflammation. 45In addition to this, HAderived biocompatible materials have also been used to repair perforated TMJ discs. 46Veronica Iturriaga et al. found that compared to HMW-HA, LMW-HA had better therapeutic effects on MIAinduced TMJOA in rabbits. 47However, the experimental results of Wen, S. et al. showed that the therapeutic effect of HMW-HA is better than LMW-HA. 48In addition, one study found that LMW-HA reduced survival in mice with post-intensive care syndrome in the short term but increased survival in the long term. 49In vitro studies, we found that LMW-HA promoted M1 macrophage polarization and the expression of ALPK1.Besides, HMW-HA had an inhibitory effect on inflammation.We consider that HMW-HA inhibits inflammation and the clinical therapeutic effects of HMW-HA are associated with inhibition of the expression of ALPK1 and M1 macrophage polarization, while LMW-HA cannot be simply defined as a pro-inflammatory factor.HA mainly functions through CD44, TLR2 and TLR4. 50Specifically, LMW-HA promotes inflammation by activating the TLR2 and TLR4 signalling pathways, 51 while its binding affinity with CD44 is lower. 52HMW-HA inhibits inflammation by regulating the TLR2, TLR4 and CD44 signalling pathways. 53,54HA may have different effects due to differences in molecular weight and microenvironment.
In summary, our results provide the initial understanding that ALPK1 regulates synovitis by promoting nuclear PKM2-mediated M1 macrophage polarization, while HMW-HA inhibited the expression of ALPK1 and M1 macrophage polarization.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.
7 cells.As shown in Figure S1, when RAW264.7 cells were treated with different rhALPK1 concentrations of 2, 5, 10, 15, 20 and 25 ng/mL, the cell viability was almost 100%, and at the concentration of 20 ng/ mL, the viability of RAW264.7 cells is closest to 100%.Based on the preliminary results, the experimental concentration of rh-ALPK1 was finally determined to be 20 ng/mL.As a possible proinflammatory molecule, does ALPK1 promote M1 macrophage in vitro?In our study, we used rhALPK1 (20 ng/mL) to stimulate RAW264.7 cells in the presence of LPS and IFNγ.The expression of INOS, CD86, TNFα, IL-6 and IL-1β was elevated in RAW264.7 F I G U R E 1 ALPK1 is highly expressed in macrophages of TMJ inflamed synovium.(A) Upregulation of ALPK1 in the synovial fluid of patients with TMJ synovitis was discovered by ELISA, as compared with patients with anterior disc displacement with reduction (ADDWR).Mann-Whitney U-test, minimum to maximum with all points, n = 8-11, p = 0.0328.(B) Haematoxylin and eosin (H&E) staining showed the control synovium and inflamed synovial.(C) The expression of ALPK1 was examined in TMJ synovium.(D) Quantitative analysis of ALPK1 in TMJ synovium.Mann-Whitney U-test, minimum to maximum with all points, n = 5, p = 0.0159.(E) The expression of ALPK1 was examined in TMJ synovial macrophages and TMJ synovial fibroblasts.(F) Quantitative analysis of ALPK1 in TMJ synovial macrophages and TMJ synovial fibroblasts.Mann-Whitney U-test, minimum to maximum with all points, n = 5, p = 0.0079.*p < 0.05.**p < 0.01.